Evidence for an induced conformational change in the catalytic mechanism of homoisocitrate dehydrogenase for Saccharomyces cerevisiae: Characterization of the D271N mutant enzyme

Abstract

Homoisocitrate dehydrogenase (HIcDH) catalyzes the NAD(+)-dependent oxidative decarboxylation of HIc to α-ketoadipate, the fourth step in the α-aminoadipate pathway responsible for the de novo synthesis of l-lysine in fungi. A mechanism has been proposed for the enzyme that makes use of a Lys-Tyr pair as acid-base catalysts, with Lys acting as a base to accept a proton from the α-hydroxyl of homoisocitrate, and Tyr acting as an acid to protonate the C3 of the enol of α-ketoadipate in the enolization reaction. Three conserved aspartate residues, D243, D267 and D271, coordinate Mg(2+), which is also coordinated to the α-carboxylate and α-hydroxyl of homoisocitrate. On the basis of kinetic isotope effects, it was proposed that a conformational change to close the active site and organize the active site for catalysis contributed to rate limitation of the overall reaction of the Saccharomyces cerevisiae HIcDH (Lin, Y., Volkman, J., Nicholas, K. M., Yamamoto, T., Eguchi, T., Nimmo, S. L., West, A. H., and Cook, P. F. (2008) Biochemistry47, 4169-4180.). In order to test this hypothesis, site-directed mutagenesis was used to change D271, a metal ion ligand and binding determinant for MgHIc, to N. The mutant enzyme was characterized using initial rate studies. A decrease of 520-fold was observed in V and V/KMgHIc, suggesting the same step(s) limit the reaction at limiting and saturating MgHIc concentrations. Solvent kinetic deuterium isotope effects (SKIE) and viscosity effects are consistent with a rate-limiting pre-catalytic conformational change at saturating reactant concentrations. In addition, at limiting MgHIc, an inverse (SKIE) of 0.7 coupled to a significant normal effect of viscosogen (2.1) indicates equilibrium binding of MgHIc prior to the rate-limiting conformational change. The maximum rate exhibits a small partial change at high pH suggesting a pH-dependent conformational change, while V/KMgHIc exhibits the same partial change observed in V, and a decrease at low pH with a pKa of 6 reflecting the requirement for the unprotonated form of MgHIc to bind to enzyme. However, neither parameter reflects the pH dependence of the chemical reaction. This pH independence of the chemical reaction over the range 5.5-9.5 is consistent with the much slower conformational change that would effectively perturb the observed pK values for catalytic groups to lower and higher pH. In other words, the pH dependence of the chemical reaction will only be observed when chemistry becomes slower than the rate of the conformational change. Data support the hypothesis of the existence of a pre-catalytic conformational change coupled to the binding of MgHIc.

Keywords: Homoisocitrate dehydrogenase; Initial rate studies; Isotope effects; Site-directed mutagenesis; Viscosity; pH-Rate profiles.

Figure 1

Alignment of the protein sequences of ScHIcSH, SpHIcDH, and TtHIcDH. The multiple sequence alignment was carried out using ClustalW2 (version 2.1) (23) and the figure was prepared using Boxshade. The black-shadowed residues are identical, while those that are gray-shadowed are conservative substitutions. The (*) shown below the alignment show likely metal ion ligands, D243, D267, D271, and the general base, K206, and general acid, Y150, residues in the ScHICDH sequence.

Figure 2

Active site of the HIcDH enzyme from S. pombe. A view of the active site with the peptide glyglygly bound in the NADP(H) site. Residues are numbered according to the SpHIcDH sequence and the ScHIcDH sequence in (). The residues with a prime are contributed by another subunit. Carbon atoms are in green for protein and yellow for the GGG ligand, N atoms are blue and O atoms are red. The general base, K196, and general acid, Y133 are shown at the top of the figure with the putative aspartate metal ion ligands below. Two arginine residues donate hydrogen bonds to two of the carboxylates of Hic are shown to the right. The figure was prepared using the PyMOL Molecular Graphics System, Version 1.7.4 Schrödinger, LLC.

Figure 3

pH dependence of V/E_t for the oxidative decarboxylation reaction of the D271N mutant of ScHIcDH with Hic as the substrate. Initial velocity data were measured at 25 °C. The points are experimentally determined values, while the curve is theoretical based on a fit of the data using eq. 4.

Figure 4

pH dependence of V/K_MgHIcE_t for the oxidative decarboxylation reaction of the D271N mutant of ScHIcDH with Hic as the substrate. Initial velocity data were measured at 25 °C. The points are experimentally determined values, while the curve is theoretical based on a fit of the data using eq. 5. The two points above the curve below pH 6 had lower precision than the rest of the data and were weighted lower for the final fit.

Scheme 1

Proposed Chemical Mechanism of ScHIcDH. I: Enzyme is in an open or partial open form upon binding of NAD⁺, and a conformational change closes the active site and positions reactants for catalysis, upon binding of MgHIc. The [ ]* reflects the closed form of the enzyme with all of the catalytic steps taking place in the closed form. II: Hydride transfer is facilitated by K206 acting as a general base. The conserved aspartate metal ion ligands are shown in this first figure only and are assumed in the remaining figues (II–V). III: Decarboxylation occurs with the metal ion serving as a Lewis acid and K206 acting as a general acid to donate a proton. IV: The general base (K206) and general acid (Y150) catalyze the enol-keto tautomerization to give α-ketoadipate (V). VI: A conformational change occurs to release α-ketoadipate.